Fungicidal toxins from biocontrol bacteria

ABSTRACT

A biocontrol bacteria, Bacillus cereus ATCC 53522, has been found to naturally synthesize two antibiotics, here designated zwittermicin A and antibiotic B. Zwittermicin A is a 396 dalton linear aminopolyol, a class of metabolite previously unknown in B. cereus. Antibiotic B is an aminoglycoside. Both antibiotics exhibit inhibitory activity on many fungal and bacterial pathogens.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agencies:

AID, Grant No.: DHR-5600-G-00-0100-00

NSF, Grant Nos.: DCB8819401; DUE-9156087

USDA, AGRICCREE 92-34103-7170; AGRICCSRS 89-37262-4746; 593-0038-04

USDA, Grant No: 89-34190-4316 (CPBR Award Nos: 593-0009-14; 593-0120-04)

USDA, Grant No: 92-34190-6941; (Purdue Agree. No.: 593-0120-04)

The United States has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.07/878,800 filed May 5, 1992, abandoned, which was acontinuation-in-part of Ser. No. 07/758,644 filed Sep. 12, 1991,abandoned, which was a divisional of Ser. No. 07/194,399 filed May 16,1988 now U.S. Pat. No. 5,049,379, which was a continuation-in-part ofSer. No. 07/077,850 filed Jul. 27, 1987, abandoned, which was acontinuation-in-part of application Ser. No. 06/890,402, filed Jul. 25,1986, now U.S. Pat. No. 4,877,738.

TECHNICAL FIELD

The present invention relates to fungicidal compounds derived frombiocontrol bacteria originally found useful in combatting damping offand root rots in plants.

BACKGROUND OF ART

Certain plants, of which alfalfa, soybeans, and common beans areexamples, suffer from disease conditions called "damping off" and "rootrot." The symptoms of damping off include the desiccation and subsequentdeath of seedlings soon after germination. Root rot symptoms includechlorosis and wilt of leaves and yellow to brown lesions with diffusemargins on roots and stems. The lesions can eventually lead to girdlingand subsequent root decay resulting in decreased robustness in the plantor even in death. Often plants suffering from root rot begin by showingsuch symptoms, which may be mistaken as symptoms of drought andstarvation. Such plants may be more vulnerable than healthy plants toattack by other pathogens, which are then mistaken as the cause of thedeath of the plants.

Damping off and root rot are merely two different sets of symptomscaused by infection of the plant by the same fungi and, in particular,by members of the Phytophthora, Pythium, Aphanomyces, Rhizoctonia, andFusarium genera. Thus, Phytophthora megasperma f. sp. medicaginis (nowformally known as Phytophthora medicaginis, and referred to hereinafteras "Pmm") causes both damping off and root rot in alfalfa when soils arewet in most parts of the world where alfalfa is grown, and Phytophthoramegasperma f. sp. glycinea has been shown to cause root rot in soybeansunder wet growing conditions. However, fungi from among the other generalisted also are believed to attack alfalfa and soybeans. Root rot incommon beans is believed caused by a complex of fungi including membersof more than one of the genera referred to.

In general, control of damping off and root rot has been attempted bybreeding for resistant plants. However, completely resistant cultivarshave not been developed such that damping off and root rot remain majorcauses of crop loss. This is especially true under chronically wetgrowing conditions or when the same crop is planted repeatedly in thesame fields. Certain fungicides such as metalaxyl partially control rootrot. However, such fungicides are fairly expensive. For some crops, suchas alfalfa, their use is not economically feasible. Also, resistance ofthe fungi to the fungicides can develop rapidly.

"Biological control" is defined as pathogen control by the use of asecond organism. Mechanisms of biological control are diverse. Forexample, certain enteric bacteria have been examined for theirusefulness in biological control of root rot in alfalfa. It is believedthat control is obtained by competition between the enteric bacteria andthe fungi for space on the surface of the alfalfa roots. In contrast, atoxin produced by one species of bacteria may be used to control anotherspecies of bacteria that appears as a pathogen. Bacterially producedantibiotics are an example of such toxins. The toxin can be isolatedfrom the species producing it and administered directly, as is thecommon procedure with penicillin, or the species itself may beadministered under appropriate circumstances to produce the toxin insitu. Once identified, such toxins produced by soil-dwelling bacteriamay have utility in diverse other areas as antifungal or antibioticagents.

BRIEF SUMMARY OF THE INVENTION

The present invention is summarized in that an antibiotic toxin has beenisolated from Bacillus cereus, the toxin being designated zwittermicinA, which is characterized and identified below.

The present invention is further summarized in the identification of asecond toxin, here designed antibiotic B, also isolated from Bacilluscereus and also characterized and identified below.

The present invention is also directed toward the use of the novelantibiotic, zwittermicin A, and antibiotic B toward the control offungicidal and bactericidal disease.

Other objects, features and advantages of the present invention willbecome apparent from the following specification.

BRIEF DESCRIPTION OF THE DRAWING FIGURE

FIG. 1 illustrates the determined chemical structure of the zwittermicinA molecule.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

An original bacterial strain was isolated from soil that exertsbiological control over species of fungi responsible for damping off androot rot in plants. The strain has been deposited in the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 US, giventhe designation ATCC 53522, and shall hereinafter be referred to as"ATCC 53522." The deposit of ATCC 53522 was made on Jul. 24, 1986. Ithas further been discovered that certain mutants of ATCC 53522 alsoprovide biological control comparable to that provided by ATCC 53522.These bacteria have been obtained in substantially pure cultures. A"substantially pure" culture shall be deemed a culture of a bacteriacontaining no other bacterial species in quantities sufficient tointerfere with replication of the culture. In addition, it has beendiscovered that the biological control is exerted by means of a toxinproduced by the disclosed bacterial strains.

ATCC 53522 and what are defined below as its "protecting" mutants,together with antibiotics produced thereby, inocula containing thebacteria or their antibiotics, and methods for protecting plants fromdamping off and root rot that utilize the bacteria or their toxins arethe subject of a co-pending patent application. Now a particularmolecule, compounds found in supernatant fluid and other bacteria-freefluid and culture medium removed from a culture of ATCC 53522 or of itsprotecting mutants, has been found to be a "protecting antibiotic," asthat term is defined below. These compounds have been so characterizedas to be identifiable independent of its source in cultures of ATCC53522, or its protecting mutants and, the two compounds shall bereferred to herein by the coined terms "zwittermicin A" and "antibioticB." Another fraction from the supernatant fluid from a culture of B.cereus ATCC 53522 has been found biologically active, having acapability to lyse Pmm zoospores, but, as revealed below, this zoolysinactive fraction does not have the antifungal activity of theantibiotics.

The method by which the biological control referred to in the precedingparagraph may be verified to exist is the "plant protection assay"detailed below. "Biological control" of fungi causing damping off androot rot shall be deemed to exist if, when an effective quantity of ATCC53522, its mutants that exhibit biological control, the antifungal toxinproduced by them, Bacillus cereus antibiotic, or any other compound ormolecule is placed in the soil or other growing medium in the immediatevicinity of the plant to be protected, a statistically significantreduction in the symptoms of damping off or root rot occurs. An"effective quantity" to combat damping off and root rot shall be thatquantity sufficient to result in such a visibly significant reduction ofsymptoms. Clearly, if no quantity of a bacteria or any toxin or othercompound is an effective quantity as so defined, that bacteria, toxin,or compound is not capable of exerting biological control over the fungicausing damping off and root rot.

ATCC 53522 and those of its mutants capable of exerting such biologicalcontrol shall sometimes be referred to collectively as "protecting"bacteria. Bacillus cereus antibiotic and other toxins capable ofexerting such biological control shall sometimes be referred to as"protecting" compounds or toxins. Plants, including seeds, seedlings,and mature plants, treated with such an effective quantity of protectingbacteria, their toxins, or Bacillus cereus antibiotic shall be referredto as "protected" from root rot or damping off.

ATCC 53522 was one of some 500 bacteria strains isolated from alfalfaroots and accompanying soil obtained from fields at the University ofWisconsin Experimental Farms at Arlington and Marshfield, Wis., and fromtwo private farms at Verona and Cross Plains, Wis. The roots were cutinto 1 cm segments, and each segment was placed in 10 ml of sterile,distilled water. The root segment and water then were sonicated at 20%maximum power with a Vibra-Cell 250 watt sonicator obtained from Sonicsand Materials, Inc., Danbury, Conn. Sonication was continued for 15seconds. The sonicated mixture then was diluted in sterile, distilledwater, and the dilutions were placed on trypticase soy agar (hereinafterreferred to as "TSA") in petri plates to form dilution plates. TSAcontains 30 g/l trypticase soy broth (hereinafter referred to as "TSB")obtained from BBL Microbiology Systems, Inc., Cockeysville, Md., and 15g/l agar. TSA and TSB are conventional bacterial culture media wellknown to those skilled in the art.

The dilution plates were incubated at 28° C. for two days. For each rootsample, bacterial colonies were selected from the dilution plate thathad the highest number of distinguishable colonies. One colony of eachvisually distinguishable morphology on the plate was sampled with asterile loop and was plated on a new TSA culture plate to allow thedevelopment of colonies in plates free from contamination by otherbacteria. After two days incubation at 28° C., a single colony wasselected from the resulting bacterial growth and was used to inoculate aTSA slant. The resulting slant cultures were stored at 4° C. until theywere screened by the plant protection assay disclosed below.

Five hundred different slant cultures were obtained by this method. As aconsequence of the isolation procedure just reviewed, it was extremelyunlikely that any of these 500 cultures were immediate siblings.However, fewer than 500 separate bacterial species were isolated. Forexample, a number of different cultures were obtained of bacteria whosecolonies had the appearance of Bacillus cereus, including the cultureidentified above as ATCC 53522. However, each of these cultures had beenobtained from a different root segment, and the root segments themselveswere obtained from fields from four different geographical locations.Consequently, the chances that a single strain was present in more thanone slant culture are very small. This fact is confirmed by theappearance of ATCC 53522 in only one of the 500 cultures.

Each of the cultured isolates that were obtained by the procedure justdescribed were screened for their ability to protect alfalfa seedlingsfrom damping off caused by Pmm. Initial screening was performed on thecultivar Iroquois, which is known to be vulnerable to Pmm. One gram ofIroquois alfalfa seeds was soaked in 18M sulfuric acid for 10 minutes.The seeds were then washed in 2 liters of sterile distilled water andwere placed in 10 ml of sterile water and shaken at 28° C. for 24 hours.Next the seed coats were removed manually with forceps, and theseedlings were planted in test tubes containing 5 ml sterile, moistvermiculite. Three seedlings were planted in each test tube. Two daysafter the seedlings were planted, each test tube was inoculated with 0.3ml of a three-day-old culture of the bacterial isolate to be tested.These cultures had been grown to saturation in TSB and had sporulated.Then each tube immediately was inoculated with 10³ zoospores of Pmm.

The Pmm zoospores had been produced by the method of S. A. Miller (1982)"Cytological and Biochemical Factors Involved in the Susceptible, HostResistant and Non-host Resistant Interactions of Alfalfa withPhytophthora megasperma," Ph.D. thesis, University of Wis. By thismethod, a sample of a colony of Pmm was transferred to an agar media onwhich it could grow. Conventional V8 media was used, consisting of 200ml V8 vegetable juice, 2.5 g CaCO₃, and 15 g agar in 800 ml water.However, any agar media such as conventional tomato juice agar or carrotagar encouraging the growth of the fungus would be sufficient. Thesample of the fungus colony was incubated at 24° C. for 4 days and thenat 28° C. for an additional 3 days. A growing colony of Pmm developed.The agar around the colony was excised to leave a section of undisturbedagar with the growing fungus on it surrounded by a "moat" formed by theexcision of agar. This moat was filled with sterile water to the levelof the agar that had not been excised. The plate was incubated at 16° C.for one hour, whereupon the water was replaced, and the plate wasincubated at 16° C. for an additional 5 hours. Zoospores were releasedfrom the fungus into the water of the moat. The concentration ofzoospores in the water was measured with a hemacytometer, and a sampleof the water was diluted with additional sterile water at 16° C. toreach a final concentration of zoospores of 10⁴ /ml.

After addition of the zoospores, the test tubes containing the plantswere incubated at 24° C. with a 12 hour photoperiod for 5 days, at whichtime the plants were evaluated for symptoms of damping off. Using Pmmand cultivar Iroquois, all control plants consistently were dead. Thus,the fact that a plant survived at all was evidence of biological controlexerted by the bacterial isolate used. All bacteria that demonstratedthat minimal amount of effectiveness for biological control wereretested by this same method to verify the consistency of such control.The screening procedure just described constitutes a particular exampleof the plant protection assay described more generally below.

Of the 500 isolates from the 4 sites in Wisconsin referred to above,only ATCC 53522 strain was identified as having the ability consistentlyto exert biological control of Pmm in Iroquois alfalfa, as evidenced byat least 20 separate experiments. The level of control was such thatalfalfa seedlings subjected to such control under the conditions of thescreening procedure were visually indistinguishable from alfalfaseedlings that had never been exposed to Pmm. ATCC 53522 has beenclassified as Bacillus cereus, based on physiological tests, its colonymorphology, and its spore size, shape, and position. Thus, ATCC 53522produces acetoin, forms an acid from glucose broth, hydrolyzes starch,and grows in anaerobic agar. These characteristics, together with colonymorphology, and spores size, shape, and position observed in ATCC 53522are cited as distinctively characteristic of Bacillus cereus by R. E.Buchanan and N. E. Gibons, co-editors (1974), Bergey's Manual ofDeterminative Bacteriology, 8th Edition, pp. 532-535.

Bacillus cereus is a not uncommon bacterium in field soils. However,strains of Bacillus cereus demonstrating antifungal activity are almostunheard of. The inventors originally tested two known strains ofBacillus cereus obtained from entirely separate sources and foundneither of them to exhibit the anti-fungal properties of ATCC 53522.Subsequently, as discussed further below, a method was derived to screenother field isolates for antibiotic production, and other such strainscan now readily be found. In the original screening, however, of the 500root-associated bacteria reviewed in the isolation process, many wereprobably Bacillus cereus and, in fact, many of them had the same colonymorphology as ATCC 53522, but none of these other strains exhibited theantifungal qualities of ATCC 53522. S. Wakayama, et al. (1984),Antimicrob. Agents Chemother., 26, 939-940, describe antifungal activityin a strain of Bacillus cereus. However, most of the antifungalantibiotics are made by Bacillus subtilis, which is easilydistinguishable from ATCC 53522. The antifungal toxin produced by ATCC53522 differs from that of the reported strain of Bacillus cereusreferred to in that the toxin is of lower molecular weight and hasdifferent solubility properties. In addition, ATCC 53522 differs fromthe reported Bacillus cereus strain in that it grows anaerobicallywhereas the reported strain does not. Consequently, it is clear that thetwo Bacillus cereus strains are not the same and that their toxins arenot the same.

The following is a disclosure of the plant protection assay whereby atest material such as a bacteria, an antibiotic, or the like, may betested for its ability to exert biological control over a fungus capableof causing the symptoms of damping off or root rot. The seed of theplant to be protected is planted in a planting medium in the presence ofdamping off or root rot causing fungi. The planting medium may be a dampsoil containing such fungi, vermiculite in water with the fungi presenteither in the vermiculite and water or in or on the seed, or any otherplanting medium in which the seed will grow and the fungi may freelydevelop. The bacteria, antibiotic, or other test material is placed atleast in the immediate vicinity of the seed. Such placement shall beunderstood to be in the "immediate vicinity" of the seed if any solubletest material or any soluble exudate of a bacteria being tested will bein actual contact with the seed as it germinates.

Preferably the seed is coated with the test material, and when the testmaterial is so used with respect to a seed, it shall be referred tohereinafter as a "seed inoculum." The process of coating seed with aseed inoculum is generally well known to those skilled in the art, andany conventional method that does not require conditions sufficientlyharsh to kill bacteria or destroy toxins or other materials included inthe seed inoculum is adequate. The plant seed to be protected isdrenched in a broth culture of the bacteria and is mixed vigorously withit to coat the surface of the seed with the bacterial suspension. Theseed may then be dried aseptically, preferably by being placed within alaminar flow hood on a sterile surface such as a sterile petri plate.The result is a dry, seed inoculum-coated seed. When the coated seed isplanted in the planting medium, the test material accompanies it toreside in the immediate vicinity of the seed.

After a time sufficient for seedling growth and the expression of thesymptoms of damping off, seedlings developing from the planted seed maybe evaluated for visual evidence of protection, when compared tocontrols. In strains of alfalfa, known to be vulnerable to damping off,2 weeks of growing time in a growth chamber at 24° C. with a 12 hourphotoperiod was found to be a period sufficient for the expression ofsymptoms of damping off when seedlings were being grown in test tubescontaining roughly 10³ to 10⁴ zoospores of Pmm or comparable, dampingoff-causing fungi. Protected seeds developed into seedlings visuallyindistinguishable from uninfected seeds while control seedlingsdeveloping from unprotected seeds were killed.

Protecting mutants of ATCC 53522 include both naturally occurring andartificially induced mutants. For example, ATCC 53522 is generallysensitive to the antibiotics rifampicin and neomycin. However, naturallyoccurring mutants of ATCC 53522 were isolated that exhibited resistanceto one or the other of these antibiotics. Certain of these mutants, aswell as one naturally occurring mutant distinguishable from the parentATCC 53522 strain by the appearance of its colonies, are discussed inthe Examples below and were found to protect alfalfa plants in the plantprotection assay. Other mutants of ATCC 53522 were artificially inducedby subjecting ATCC 53522 to the mutagen N-methyl-nitrosoguanidine inconventional ways, as is discussed in the Examples below. Most of theseinduced mutants also were found to protect alfalfa plants in the plantprotection assay.

Various mutagenesis studies have also been done on cultures of ATCC53522 which have resulted in mutant colonies which are deficient inproduction of the antibiotics and which are deficient in biocontrolactivity, as determined by the biocontrol assays described herein. Thosemutant colonies also were deficient in antibiotic production. Ananalysis of the mutant colonies for both biocontrol activity and forantibiotic accumulation revealed that the interpretation that thebiocontrol activity was associated with the accumulation of bothantibiotics as zwittermicin A and antibiotic B was consistent with thedata uncovered from the mutant strains. Residual disease suppressiveactivity was detected in some strains that lack detectible antibioticproduction, and such suppressive activity may be due to thezoospore-lysis activity or to another agent. This observation isconsistent with the fact that many biocontrol bacterial colonies maydepend on multiple strategies for disease suppression and the data wouldthus suggest that the antibiotics are required, but not sufficient, forthe full biological control of cultures. Nevertheless, the antibioticsmay have independent utility in other environments for the control ofbacterial or fungicidal agents, as described in more detail below.

As has been disclosed above, it has been further discovered that activeanti-root rot toxins, identified herein as the B. cereus antibioticszwittermicin A and antibiotic B, are produced by ATCC 53522 and those ofits mutants that are characterized by their abilities to protect plantsfrom root rot in the plant protection assay. The two Bacillus cereusantibiotics may be collected from growth media in which the bacteriahave been cultured and has been prepared in a substantially pure form. Apreparation of Bacillus cereus antibiotic shall be deemed "substantiallypure" if it is sufficiently free of interfering substances as to be ableto be active to inhibit root rot by Pmm. The two Bacillus cereusantibiotics are effective to protect plants from damping off and rootrot, even when separated from the bacteria producing it and applied toseed and to seedlings that have been placed in a planting mediumcontaining root rot causing fungi. As is discussed below, theeffectiveness of the application of two Bacillus cereus antibiotics isdemonstrable by the plant protection assay, with the antibiotic beingsubstituted for a protecting bacteria. Thus, the invention includeseither or both antibiotics and a seed inoculum containing effectivequantities of either Bacillus cereus antibiotic.

As has been disclosed above, the Bacillus cereus antibiotics may beisolated from ATCC 53522 and its protecting mutants by filtering thebacteria from the culture media in which they have been grown to asporulated culture. Other conventional purification and concentrationsteps may then be undertaken as may be considered convenient ordesirable, so long as the toxin remains active, as may be demonstratedby the plant protection assay.

Both of the antibiotics from Bacillus cereus described herein can bereadily isolated from cultures of Bacillus cereus ATCC 53522 by cultureof the bacteria and concentration of the resulting antibiotics from theculture supernatant. The supernatant can be fractionated in a column andthen separated by electrophoresis to identify fractions which exhibitthe biocontrol activity. It has been found that both antibiotics willstain with ninhydrin or silver nitrate.

The chemical formula and structure of the antibiotic zwittermicin A hasbeen studied extensively. The molecular weight of a molecule, which wasoriginally identified as Bacillus cereus antibiotic, was originallythought to be between 500 and 1000 daltons. Subsequent measurement ofthe molecular weight of purified antibiotic, now referred to azwittermicin A, by mass spectroscopy revealed a molecular weight ofabout 396 daltons. The zwittermicin A antibiotic is soluble in methanoland insoluble in acetone, chloroform, and ethyl acetate. The antibioticzwittermicin A moves as both an anion and a cation in an electric field,although it is a very weak anion. In repeated experiments, theantibiotic zwittermicin A has been tested for various protectingabilities by plant protection assays. The tests, some of which arediscussed below, have revealed that the zwittermicin A antibiotic hasproven useful antibiotic capabilities against Phytophthora and Pythiumspecies as well as other fungal species. In addition, the antibioticzwittermicin A also inhibits growth of some bacteria, notably Erwiniaherbicola, several Pseudomonas species, and some E. coli strains.

The chemical structure for the zwittermicin A molecule has beententatively identified. Shown herewith in FIG. 1 is the determinedchemical structure of the molecule which has been identified. Themolecule is an aminopolyol antibiotic which represents a new class ofantibiotics isolated from Bacillus species. Zwittermicin A is a veryweak acid, and will migrate as an anion at pH 9.2 as did otheramide-containing compounds. It is appropriate to rely on biologicalactivity as a detection method during purification or fractionationbecause the antibiotic zwittermicin A does not contain chromophores thatcan be detected spectrophotometrically.

Structure illustrated on FIG. 1 was determined by NMR and massspectrometry studies of the native molecule, the acetylated derivative,and a hydrolysis product. While it is believed that this structure iscorrect, it is yet possible that there might be minor misplacements ofsmaller residues without affecting the overall chemical characterizationor structure of the molecule. It is known, as described above, that themolecule is certainly a linear aminopolyol, a cation, and a very weakacid. The structure disclosed in FIG. 1 is consistent with thatinterpretation and is believed to be the correct molecular structure.

At this point, an incomplete structure is known for the antibiotic B.The antibiotic B also shows inhibitory activity in a biocontrol assay.The antibiotic B will also bind to CM-sephadex or amberlight IRC-50 atpH 7.0 and will elute when the pH is raised above 10.0. The stainingproperties and nuclear magnetic resonance profile of antibiotic Bindicates that it is an aminoglycoside. While the antibiotic B has aslightly narrower spectrum of antifungal and antibacterial activity thanzwittermicin A it nevertheless exhibits reasonably broad antifungal andantibacterial efficacy.

As the examples below indicate, both zwittermicin A and antibiotic Bhave significant activity against a variety of pathogenic fungi andbacteria. As the data also indicate, not only are the antibiotics usefulseparately, they also have certain synergistic activities when used incombination with each other. It is believed that the antibiotics can beprepared by culture of the Bacillus cereus ATCC 53522, but can equallybe recovered from a wide variety of other Bacillus cereus cultures.Described below is a plant protection assay developed to test plantprotection activity as initially isolated using ATCC 53522. It isbelieved that that same assay can be used to test other candidateBacillus cereus strains for biocontrol activity and it is believed thatthose strains would also make one or both of the antibiotic zwittermicinA or antibiotic B described herein.

The antibiotics may readily be isolated from colonies of bacteria whichproduce them, such as ATCC 53522 by recovering the supernatant fromsporulating colonies of the microorganisms. The supernatant, asdescribed above, can be fractionated in a column and then separated byelectrophoresis to identify the fractions which exhibit the biocontrolactivity. Using high voltage paper electrophoresis, the two moleculesidentified herein as zwittermicin A and antibiotic B can readily berepeatably recovered. At pH 9.2, the high voltage paper electrophoresis(HVPE) yields 2 spots which are associated with biocontrol activity. Thefirst spot, associate with zwittermicin A, had a relative mobility(R_(f)) of 0.30 compared with orange G. The antibiotic B spot had anR_(f) of 0.032. If the same HVPE process is conducted at pH 1.7, therelative mobilities of zwittermicin A and antibiotic B are R_(f) -1.042and -0.909 respectively.

As described below, both zwittermicin A and antibiotic B have a broadantibiotic activity against many fungal, and also some bacterial,pathogens. The activity extends not only to plant pathogens, but also topotential mammalian pathogens. It is also revealed from the data below,the level of toxicity for each individual pathogen varies over asignificant range. Accordingly, the significant amount necessary tocontrol a particular pathogen can be determined empirical by in vitrostudies of the type described below. Based on such studies an "effectiveamount" can be determined for a particular target organism.

EXAMPLE 1 Plant Protection Assay of ATCC 53522 Using Alfalfa

The screening procedure disclosed above was repeated as an applicationof the plant protection assay to test the protective ability of ATCC53522 with alfalfa. The cultivar of alfalfa used was Iroquois. Thefungus used was Pmm. One gram of seeds was soaked in 18M sulfuric acidfor ten minutes, washed in 2 l of sterile distilled water, placed in 10ml of sterile distilled water, and shaken at 28° C. for 24 hours.Thereafter, the seed coats were removed with forceps, and the seedlingswere planted in test tubes containing 5 ml of moist vermiculite. Threeseedlings were planted in each test tube. After two days, each test tubewas inoculated with 0.3 ml of a three day old culture of ATCC 53522 thathad been grown in TSB to saturation. Thereafter, each tube wasinoculated with 10³ zoospores of Pmm. The plants then were incubated at24° C. with a 12 hour photo period for 7 days, whereupon the plants wereevaluated for viability. All of the control seedlings were dead. Most ofthe seedlings that had been treated with ATCC 53522 had the appearanceof normal seedlings that had not been exposed to Pmm.

EXAMPLE 2 Plant Protection Assay of ATCC 53522 with Soybeans

The procedure of Example 1 was repeated with soybeans of the varietyMcCall substituted for the alfalfa seeds and zoospores of Phytophthoramegasperma f. sp. glycinea substituted for the zoospores of Pmm. Insteadof being planted in test tubes, the soybean seeds were planted in 10 mlplastic cones having holes in the bottom, and the cones were placed in apan of water. The seedlings were examined for protection two weeks afterinoculation with the zoospores. Ten out of 10 controlled seedlings werekilled by the fungus. All of the seedlings that had been treated withATCC 53522 survived with healthy, white roots.

EXAMPLE 3 Field Test of ATCC 53522

Alfalfa seeds of the cultivar Iroquois were mixed in a suspension ofATCC 53522 in 1.5% methyl cellulose. The bacteria had been cultured on aTSA plate that had been incubated at 30° C. for three days, by whichtime the culture had sporulated. The culture then was scraped into 3 mlof the 1.5% methyl cellulose solution to provide the suspension ofbacteria. One gram of alfalfa seeds was added to this suspension and wasmixed thoroughly therewith. The seed then was spread on sterile petriplates and dried overnight in a laminar flow hood. The coated seeds wereplanted in circular plots 0.3 m in diameter at Marshfield, Wis. Owing todry growing conditions, both emergence of plants and evidence of Pmmdamping off were poor. Nevertheless, emergence in a control, untreatedplot was 18% whereas in the plot planted with bacterium-treated seed,emergence was 30%. An additional plot was planted with seed that hadbeen coated with a fungicide, metalaxyl, a conventional control agentfor damping off. In that plot, emergence was 29%. Thus, it is apparentthat ATCC 53522 can protect alfalfa in the field as effectively as doesmetalaxyl. Furthermore, symptoms of root rot became apparent in thecontrol plot having untreated seeds as the growing season proceeded. Nosymptoms of root rot appeared in the plot planted with the seeds coatedwith ATCC 53522.

EXAMPLE 4 Plant Protection Assay of ATCC 53522 Toxin

The method of Example 1 was repeated with ATCC 53522 being replaced witha filtrate of a culture of that bacterium. The filtrate was prepared bycentrifuging a two day old, saturated broth culture at 10,000 g for tenminutes and then filtering the resulting supernatant twice through 0.45μfilters. The filtrate was stored at -20° C. before being applied in theplant protection assay identically to the way the bacteria had beenapplied in the experiment reported as Example 1. The protective effectobserved in treated alfalfa seedlings versus untreated seedlings wasidentical to that reported in Example 1. The filtrate used in thisexample contained Bacillus cereus antibiotic.

EXAMPLE 5 Spontaneous Mutants of ATCC 53522

Spontaneously developing antibiotic resistant mutants of ATCC 53522 wereisolated by plating a culture derived from a colony of ATCC 53522 onmedia containing an antibiotic to which ATCC 53522 normally issensitive. Several resistant colonies developed. They were each sampledwith a sterile toothpick and replated on the antibiotic-containingmedia. The mutants were then tested in the plant protection assay by theprocedure described in Example 1. Five mutants were developed that wereresistant to rifampicin. A sixth mutant was developed that was resistantto neomycin. Each of the mutants protected alfalfa plants in the plantprotection assay as applied in Example 1 as effectively as did ATCC53522.

EXAMPLE 6 Induced Mutants of ATCC 53522

A culture of vegetatively growing cells of ATCC 53522 was prepared anddiluted to a density of 10⁸ cells/ml. A quantity of this culture wastreated by exposure to 1 μg/ml N-methyl-nitrosoguanidine for thirtyminutes at room temperature. The cells then were washed with water anddilution plates were prepared on TSA. The treatment withN-methyl-nitrosoguanidine had killed 99% of the bacteria in the originalculture. Thus, the remaining viable bacteria each had a high probabilityof containing at least one mutation. Of 500 such bacteria derived fromindependent colonies, 490 were able to protect alfalfa plants againstPmm when tested by the method of Example 1.

EXAMPLE 7

The assay procedure of Example 1 was again used to demonstrate that theplant protection activity resides with the Bacillus cereus antibiotic bytesting filtrate fraction activity with the natural strain andantibiotic deficient mutants. Strain T30 is such an antibiotic deficientmutant derived from Bacillus cereus ATCC 53522. The results of thisprocedure are demonstrated in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Plant Survival                                                                Treatment           Alfalfa Tobacco                                           ______________________________________                                        None                0/18    0/12                                              ATCC 53522          18/18   12/12                                             ATCC 53522 filtrate 18/18   12/12                                             ATCC 53522 500-1000 fraction                                                                      18/18   12/12                                             T30                 0/18    2/12                                              T30 filtrate        0/18    1/12                                              T30 500-1000 fraction                                                                             0/18    0/12                                              ______________________________________                                    

This demonstrates that the plant protecting activity is in the Bacilluscereus antibiotic independent of the bacteria, and that the activity isabsent in antibiotic deficient mutants.

EXAMPLE 8 Isolation of Phage P7

A culture of ATCC 53522 was grown in tryptic soy broth with vigorousagitation. During the log-phase growth phase of the bacteria, mitomycinC was added to the media to a final concentration of 1 μg/ml. Thebacteria in culture lysed 8 to 9 hours after the addition of themitomycin C. Phage particles were isolated from the remaining culture byplating aliquots of the culture on a lawn of ATCC 53522 grown in softagar (0.4%). Individual plaques were then picked and replated again intoa similar subculture. The P7 phage has been propagated by platingsufficient lysate on a culture of a ATCC 53522 on a soft agar overlay toresult in clearing of the overlay. The overlay has been typicallyremoved from the plate, the agar removed by centrifugation, and thesupernatant stored for future use. Later, the supernatant wasreinoculated onto culture to continue to propagate and isolateadditional phage P7. Samples of the phage P7 have been deposited withthe ATCC as Accession No. 75237.

EXAMPLE 9 Isolation of Other Biocontrol Bacteria

Additional populations of Bacillus cereus were recovered from soil orfrom field-grown soybeans and from alfalfa, soybeans and snapbeansplants grown in field soils in the growth chamber. The samples weredilution plated by sonicated samples of soil, seeds, cotyledons,radicles or 1 to 2 cm root segments taken from 0-1, 0-2, 2-3, 4-5, or9-10 cm below the crown, or from the last cm of the root. For thispurpose, the crown was defined as the part of the plant at the soil-airinterface which was marked on each plant as it was removed from thesoil. Plant materials placed at either 5 or 10 ml of sterile distilledwater, which was then sonicated for 15 seconds at 20% output with a 250W Vibra-cell sonicator (Sonics and Materials) and then serially dilutedin sterile distilled water. Aliquots (0.1 ml) of the dilutions were thenplated onto a semi-selective medium. The semi-selective medium (Min IC)medium was used because few non-Bacillus bacteria will grow on it,thereby semi-selecting for the detection of Bacillus cereus. The Min ICmedium contained, per liter, 2.0 g of (NH₄)₂ SO₄, 6.0 g of KH₂ PO₄, 14.0g of K₂ HPO₄, 0.2 g of MgSO₄ --7H₂ O, 0.25 mg of MnSO₄ --H₂ O, 1.0 g oftrisodium citrate-2 H₂ O, 0.1 g of thiamine hydrochloride, 2.0 g ofL-glutamic acid, and 5.0 g of acid-hydrolyzed casein (Sigma). Afterautoclaving, 10 ml of a sterile 50% (wt/vol) glucose solution and 10 mlof sterile FeCl₃ --6H₂ O (4.0 mg/ml) were added. The Min IC medium wasalso inoculated with 12.5 micrograms/ml polymyxin B-sulfate, 50 μg/mlampicillin, and 100 μg/ml cycloheximide. The B. cereus isolates thatwere collected from the field were screened on a semi-selective media.Colonies of B. cereus were identified by their distinctive colonymorphology, i.e., large, flat, wrinkled, cream or orange coloredcolonies, on the semi-selective medium.

EXAMPLE 10 Screening of Putative Biocontrol Agents

The biocontrol agents isolated in this fashion were then subjected to aselection criteria based on the use of three assays which have beenfound to have strong correlations with each other, and which are capableof identifying Bacillus cereus strains which are capable of biocontrolactivity and which produce the zwittermicin A toxin. One assay is basedon the susceptibility of the candidate strain to infection by the P7phage. The second test is based on a laboratory biocontrol study usingErwinia herbicola. The third study is an actual stain assaying for theproduction of the zwittermicin A toxin itself. All of these assayscorrelate well, although not perfectly. So far, however, every strainexcept one found to be susceptible to P7 has tested positive for Erwiniainhibition and has stained for antibiotic production. The results ofthese assays, done collectively or singly, may be verified by biocontrolstudies on actual plants.

The phage sensitivity selection was done using the following protocol.High titer preparations (in excess of 10⁹ pfu/ml) of phage P7 wereprepared either from infected broth cultures or from top agar overlaysof Bacillus cereus ATCC 53522 as described in the prior example. Thecells were removed by centrifugation and the supernatants were filtered(0.2 micron or 0.45 micron filters). The phage preparations were titeredand stored in a refrigerator. Separately, cultures of the candidateorganisms were grown on 50% trypsin soy agar (TSA). The growth isscraped from the culture plate, suspended in a small volume of 50%trypsin soy broth (TSB), and added to three milliliters of molten 50% TStop agar (0.4% agar) and spread on a plate of 50% TSA. Drops of the hightiter phage stock, approximately 10 microliter in size, were placed onthe plate. The plates were incubated overnight at 28° C. If the drop ofphage introduced into the culture caused a clear zone, the strain wasscored as sensitive to the phage.

The laboratory biocontrol assay for Erwinia herbicola inhibition wasconducted as follows: The Erwinia culture was grown in 50% TSB withshaking, over night, at 28° C. The Erwinia cells were allowed to settleto the bottom of the tube and the stock of Erwinia was stored in therefrigerator, sometimes for as much as two weeks. The candidate B.cereus strain to be tested was grown in 50% TSB, with shaking, at 28°C., for two to three days. Fifteen microliters from the top of theErwinia stock tube, taken without shaking the tube, was placed in 1milliliter of sterile water. Eighty-five microliters of the Erwiniadilution was then spread on water agar or 25% tryptic soy agar in aplate. Four holes were cut in the plate with a sterile cork borer.Approximately 100 microliters of the candidate B. cereus test culturewas added to each of the holes cut in the plate. The zones of inhibitionof Erwinia growth around the B. cereus cultures were scored in two tothree days. Candidates were scored as positive if a zone of inhibitionappeared.

To assay for the production of the zwittermicin A toxin, cultures of thecandidate B. cereus cultures were maintained under conditions describedabove. The cultures were fully sporulated and centrifuged to removespores. The supernatant was applied to a CM Sephadex cation exchangecolumn in the ammonia form. The column was then washed with buffer (6mil 10 mM N,N bis (2-hydroxyethyl) 2-amino ethane sulfonic acid, pH7.0). The bound toxin, if present, was eluted with 10 mM3-cyclohexylamino propane sulfonic acid, pH 10.4. Fractions werecollected, dried in a rotary evaporator and resuspended in water.Resuspended material was spotted onto filter paper and subjected topreparative high voltage paper electrophoresis at pH 1.7 and 300 voltsfor 15 minutes. Filter paper that had been subjected to electrophoresiswas stained by dipping in a solution containing 0.25% ninhydrin inacetone. The plates of paper were dried and heated at 110° C. untilspots were visible. The occurrence of ninhydrin staining spots verifiedproduction of the antibiotic.

The following Table 2 summarizes the results of assaying the isolatedstrains. The results demonstrate that the three laboratory tests, P7susceptibility, Erwinia inhibition, and antibiotic detection correlatenicely with each other and with biocontrol activity. While some strainsmay fail one of the tests and still have biocontrol capability, so fareach strain that has passed one or more assay has exhibited biocontrolactivity. Hence these assays, singly or collectively, provide usefullaboratory tools to select new biocontrol strains.

                  TABLE 2                                                         ______________________________________                                        Correlation between Phage Sensitivity, Antibiotic                             Production and Biocontrol Activity for                                        B. cereus isolates                                                                       P7      Erwinia   Antibiotic                                                                           Biocontrol                                Strain     Assay   Assay     detected                                                                             Activity                                  ______________________________________                                        ATCC 53522 +       +         +      +                                         Laboratory -       -         -      nt                                        Strains                                                                       (7 strains)                                                                   Soil Isolates                                                                 1          +       +         +      +                                         3 other    -       -         nt     nt                                        strains                                                                       86 strains -       nt        nt     nt                                        Soybean Root                                                                  Isolates                                                                      1          +       +         +      +                                         5 other    -       -         nt     nt                                        strains                                                                       39 other   -       nt        nt     nt                                        strains                                                                       Alfalfa Root                                                                  Isolates                                                                      1          +       +         +      +                                         2          +       +         +      +                                         3          +       +         +      +                                         8 strains  -       +         +      +                                         1 strain   -       -         -      +                                         11 strains -       -         -      -                                         154 strains                                                                              -       -         nt     nt                                        ______________________________________                                         nt = not tested                                                          

EXAMPLE 11

A B. cereus ATCC 53522 cultures were grown in half-strength trypticasesoy broth (TSB). Then 1 liter, three-day old fully sporulated cultureswere centrifuged to remove spores and the culture supernatant wereadjusted to pH 7.0 with 2.0M NaH₂ PO₄ and then applied to a column (2.2cm×30.0 cm) containing Amberlite IRC-50 (Sigma). The column was washedwith 5.0 mM NH₄ HPO₄ /NH₃ pH 7.0 and eluted with 1.0M NH₃ pH 11.2. Thematerial in approximately 30 ml was collected from the column after thepH was raised above 10, dried in an evaporator, and quantified.

The active fractions were then purified by high voltage electrophoresis(HVPE) at pH 9.2. From the electrophoretograms, two spots wereidentified with the two antibiotics, zwittermicin A and antibiotic B.The spot for zwittermicin A had a relative mobility (R_(f)) of 0.30compared to Orange G. The spot for antibiotic B had an R_(f) of 0.032.These spots were eluted and subjected to HVPE at pH 1.7, at which theantibiotics exhibited mobilities similar to a di-cation (R_(f) =-1.042for zwittermicin A and R_(f) =-0.909 for antibiotic B).

Separately, a panel of bacterial and fungal pathogenic strains werecollected for use in an assay with purified zwittermicin A andantibiotic B to test antibiotic activity. The bacterial strains were alltested in Muller-Hinton (MH) broth (Sigma) at pH 7.3 and MH brothbuffered with 3-(n-morpholino) propanesulfonic acid (MOPs) to pH 8.0.The susceptibility testing of the Rhizobium and Lactobacillus strainswere conducted in L-broth (Maniatis, 1982). Other strains requiringspecial treatment were the Rhodosprillum which was grown in MH broth towhich 1 μg/ml of biotin was added, and the Clostridium which was grownin MH broth to which was added 20 μg/ml of sucrose.

The susceptibility testing of the fungal strains described below asconducted on potato dextrose agar (PDA) at pH 5.6 and PDA buffered withMOPs to pH 7.0.

For the bacterial susceptibility testing, the minimum inhibitoryconcentration (MICS) were determined by a broth dilution procedure. Theinocula were prepared from fresh broth cultures and diluted to provideinoculum concentrations of approximately 5×10⁵ CFU/ml. Antibiotics wereadded in two-fold dilutions, ranging from 50 μg/ml to 400 μg/ml and eachtest tube contained 1 ml. The cultures were incubated at 28° C. withshaking for 24 h, (48 h for Lactobacillus acidophilus, Streptomycesgriseus, Rhizobium meliloti, Rhizobium tropici, Rhodobacter sphaeroides,and Rodosprillum rubrum). Clostridium pasteurianum was tested underanaerobic conditions by overlaying the culture with 3 ml of sterilemineral oil and then growing the culture for 4 days at room temperature.MICs were interpreted as the lowest antibiotic concentration thatprevented visible growth. Bacteria with MICs of 50 μg/ml forzwittermicin A were retested at 0 to 50 μg/ml antibiotic in 10 μgincrements. Minimal bactericidal concentrations (MBCs) were determinedfor each bacterial strain by spreading 0.1 ml from each test culturewithout visible growth onto Muller-Hinton agar (Sigma Chemicals) plates.The plates were scored for bacterial growth after incubation at 28° C.for 24 to 48 h.

The zoosporic fungi were tested as follows: Zoospores of Aphanomyceseuteiches (2×10⁴), Phytophthora medicaginis (5×10⁴), Pythiumaphanidermatum (1×10³) and Pythium torulosum (1×10³) were spread ontoPDA plates. A well was cut into the center of the agar with a sterilizedcork borer. Purified antibiotic was pipetted into the well and theplates were incubated at room temperature for 48 h. Zones of inhibitionwere measured from the well to visible growth. MICs in this assay werethe lowest antibiotic concentration that resulted in a zone ofinhibition. Candida utilus, Saccharomyes cerevisiae and Ustilago maydiswere tested by the same procedure and approximately 1×10⁴ CFUs werespread on the PDA plates. Venturia inaequalis was tested by mixing 2×10⁵conidia into 25 ml of 50% PDA. The agar was vortexed briefly and thenpoured into a petri plate. A well was cut into the plate for theplacement of antibiotic. The other fungi were tested for antibioticsusceptibility as follows: PDA plates were incubated with a plug ofmycelia in the center of the plate. A well was cut into the agar 5 to 10mm from the plug. Purified antibiotic (200) μg, was placed in the welland the plates were incubated at room temperature. Because of thevariable growth rates of the test fungi, the plates were scored forgrowth after 2 to 6 days.

The data from the in vitro testing of antibiotic zwittermicin A andantibiotic B against bacteria is listed on Table 3 below while theresults obtained from the testing against fungal pathogens is listed onTable 4 below. Also shown below, in Table 5, is an experiment conductedin which the combined activity of zwittermicin A and antibiotic B wasevaluated against E. coli, at various combinations of each of the twoantibiotics.

                  TABLE 3                                                         ______________________________________                                        In vitro activities of zwittermicin A                                         and antibiotic B against bacteria                                                                 MIC(μg/ml).sup.a                                       Bacteria tested       ZmA     Ant B                                           ______________________________________                                        Agrobacterium tumefaciens A759                                                                      40      >400                                            Bacillus cereus 569   >400    >400                                            Bacillus cereus UW85  >400    >400                                            Bacillus cereus BAR145                                                                              >400    >400                                            Bacillus subtilis 168 >400    >400                                            Bacillus thuringiensis 4A9                                                                          >400    >400                                            Bacillus thuringiensis 4D6                                                                          >400    >400                                            Bradyrhizobium japonicum USDA 110                                                                   100     >400                                            Clostridium pasteurianum 5002                                                                       >400    >400                                            Cytophaga johnsonae 9408                                                                            >400    300                                             Escherichia coli K37  100     >400                                            Erwinia carotovora 8064                                                                             40      >400                                            Erwinia herbicola IRQ >400    >400                                            Erwinia herbicola LS005                                                                             50      400                                             Klebsiella pneumoniae 8030                                                                          200     >400                                            Lactobacillus acidophilus 4003                                                                      100     >400                                            Pseudomonas aeruginosa 9020                                                                         >400    >400                                            Pseudomonas fluorescens 9023                                                                        >400    >400                                            Rhizobium tropici CIAT 899                                                                          100     >400                                            Rhizobium meliloti 1021                                                                             50      >400                                            Rhodobacter sphaeroides 9502                                                                        50      400                                             Rhodosprillum rubrum 9405                                                                           50      >400                                            Salmonella typhimurium LT2                                                                          100     >400                                            Staphylococcus aureus 3001                                                                          200     400                                             Streptomyces griseus 6501                                                                           400     >400                                            Vibrio cholerae F115A 400     >400                                            ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        In vitro activities of zwittermicin A                                         and antibiotic B against various fungi                                                                Inhibition.sup.a                                      Fungi tested  Disease incited ZmA    Ant B                                    ______________________________________                                        Alternaria alternata                                                                        Leaf blight on beet                                                                           +      +/-                                      Alternaria tagetica                                                                         Leaf and petal blight                                                                         +      +/-                                      Aphanomyces euteiches                                                                       Seedling blight of                                                                            +      +                                        WI-98         alfalfa                                                         Aspergillus flavus                                                                          Non-pathogenic  -      -                                        Botrytis cinerea                                                                            Molds and rots of                                                                             +      -                                                      stored fruits and                                                             vegetables                                                      Candida utilus                                                                              Non-pathogenic  +      -                                        Colletotrichum phomoides                                                                    Anthracnose of tomato                                                                         +/-    -                                        Colletotrichum trifolii                                                                     Anthracnose of  +      -                                        SMM           alfalfa                                                         Cytospora cineta                                                                            Branch canker of                                                                              +      -                                                      fruit trees                                                     Drechslera poae                                                                             Leaf spot/foot rot                                                                            +      +/-                                                    of grasses                                                      Epicoccum nigrum                                                                            Leaf spot of magnolia                                                                         +      -                                        Fusarium oxysporum                                                                          Vascular wilt of tomato                                                                       -      -                                        f. sp. lycopersici                                                            Fusarium sporotrichioides                                                                   Blight of barley and                                                                          +      +/-                                                    sunflower                                                       Fusarium solani                                                                             Root rot of bean                                                                              +      -                                        Helminthosporium                                                                            Leaf spot and ear                                                                             +      +/-                                      carbonum      rot of corn                                                     Helminthosporium sativum                                                                    Foot rot of grasses                                                                           +      +/-                                      Ophiostoma ulmi                                                                             Dutch elm disease                                                                             +/-    -                                        Phoma obscurans                                                                             Leaf spot of strawberry                                                                       +      -/-                                      Phytophthora medicaginis                                                                    Root rot of alfalfa                                                                           +      +                                        Pythium torulosum                                                                           Damping-off of tobacco                                                                        +      +                                        Pythium aphanidermatum                                                                      Root rot of vegetables                                                                        +      +                                        Rhizoctonia solani                                                                          Root rot of fruits/                                                                           +      -                                        (AG1, AG4)    vegetables                                                      Saccharomyces cerevisiae                                                                    Non-pathogenic  -      -                                        Sclerotinia homoecarpa                                                                      Dollar spot of turf                                                                           -      -                                        Sclerotinia sclerotiorum                                                                    Rots of most crops                                                                            +      -                                        Typhyla incarnata                                                                           Snowmold of turf/grasses                                                                      -      -                                        Ustilago maydis                                                                             Smut of corn    +      +                                        Venturia inaequalis                                                                         Scab of apple   +      +                                        Verticillium dahliae                                                                        Wilt of potato  +/-    -                                        Verticillium albo-atrum                                                                     Wilt of alfalfa +/-    -                                        ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Combined activity of zwittermicin A                                           and antibiotic B against E. coli                                                        Zwittermicin A (μg/ml)                                                     0     10    15     20   25   30   40                                ______________________________________                                        Antibiotic B                                                                           0      +++     +   +    +    +    +    -                             (μg/ml)                                                                             50     +++     +   +    +    -    -    -                                      100    ++      +   +    -    -    -    -                                      200    ++      -   -    -    -    -    -                                      300    ++      -   -    -    -    -    -                                      400    -       -   -    -    -    -    -                             ______________________________________                                         Growth of E. coli strain K37 ranged from saturated cultures (+++) to no       visible growth (-).                                                      

As the above data demonstrates, the antibiotics have broad spectrumactivity against a variety of fungal pathogens and also have significantactivity against many bacterial pathogens. In addition, from the E. colistudy, it appears that the action of the antibiotics is synergistic andthat they act in concert to achieve levels of inhibition that neitherwould achieve alone.

What is claimed is:
 1. A compound having the following formula: ##STR1##2. The compound of claim 1 wherein the compound is produced by Bacilluscereus strain ATCC
 53522. 3. A composition of matter consistingessentially of an isolated compound having the following formula:##STR2## and an agriculturally or pharmaceutically acceptable carrier.